Structural Requirements for Multimerization of the Pathogen Receptor Dendritic Cell-specific ICAM3-grabbing Non-integrin (CD209) on the Cell Surface

Abstract

The myeloid C-type lectin dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN, CD209) recognizes oligosaccharide ligands on clinically relevant pathogens (HIV, Mycobacterium, and Aspergillus). Alternative splicing and genomic polymorphism generate DC-SIGN mRNA variants, which have been detected at sites of pathogen entrance and transmission. We present evidence that DC-SIGN neck variants are expressed on dendritic and myeloid cells at the RNA and protein levels. Structural analysis revealed that multimerization of DC-SIGN within a cellular context depends on the lectin domain and the number and arrangement of the repeats within the neck region, whose glycosylation negatively affects oligomer formation. Naturally occurring DC-SIGN neck variants differ in multimerization competence in the cell membrane, exhibit altered sugar binding ability, and retain pathogen-interacting capacity, implying that pathogen-induced cluster formation predominates over the basal multimerization capability. Analysis of DC-SIGN neck polymorphisms indicated that the number of allelic variants is higher than previously thought and that multimerization of the prototypic molecule is modulated in the presence of allelic variants with a different neck structure. Our results demonstrate that the presence of allelic variants or a high level of expression of neck domain splicing isoforms might influence the presence and stability of DC-SIGN multimers on the cell surface, thus providing a molecular explanation for the correlation between DC-SIGN polymorphisms and altered susceptibility to HIV-1 and other pathogens. Dendritic cells (DCs)4 link the innate and adaptive branches of the immune response by virtue of their capacity to recognize pathogen-specific structures (1) via pathogen-associated molecular pattern receptors (2). Immature DCs express a number of lectins and lectin-like molecules, which endow them with a broad capacity for pathogen recognition, as they mediate the specific recognition of parasitic, bacterial, yeast, and viral pathogens (3, 4). Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN, CD209) is a type II membrane C-type lectin (5, 6) abundantly expressed in vivo on myeloid DC and macrophage subpopulations (5-12), as well as on in vitro generated monocyte-derived dendritic cells (MDDCs) and alternatively activated macrophages (12-14). DC-SIGN binds a large array of pathogens, including HIV (15), Ebola (16), hepatitis C (17-19), and Dengue virus (20) and Leishmania amastigotes and promastigotes (21, 22), Mycobacterium tuberculosis (23, 24), Aspergillus fumigatus (25), and Candida albicans (26) via mannan-and Lewis oligosaccharides-dependent interactions (27,28). In addition, DC-SIGN appears to mediate DC contacts with naïve T lymphocytes through its recognition of ICAM-3 (6), DC trafficking through interactions with endothelial ICAM-2 (8), and DC-neutrophil interactions by interacting with the CD11b/CD18 integrin (29).Structurally, DC-SIGN contains a carbohydrate-recognition domain, a neck region composed of...

Enhanced receptor–clathrin interactions induced by N -glycan–mediated membrane micropatterning

“…1B). As a control for impaired nanoclustering, we include the spot size distributions obtained for a DC-SIGN mutant lacking the neck region (ΔRep), known to abrogate nanoclustering (23,27). Interestingly, the spot size distributions for wtDC-SIGN and N80A are similar, with average values around 160 nm, in agreement with previously reported wtDC-SIGN values (21,23) and significantly larger than for ΔRep.…”

“…Because DC-SIGN forms nanoclusters on the cell surface and has a single N-glycosylation site (19,(21)(22)(23), we first investigated the role of N-glycosylation on the nanoscale organization of DC-SIGN using stimulated emission depletion (STED) nanoscopy. We used CHO cells stably expressing wild-type DC-SIGN (wtDC-SIGN) and a receptor variant presenting a point mutation within the DC-SIGN N-glycosylation motif (denoted as N80A), known to prevent receptor glycosylation (27). This cell system recapitulates DC-SIGN essential activities, such as antigen binding, internalization, and trafficking (23)(24)(25).…”

A role of the Ca 2+ binding site of DC-SIGN in the phagocytosis of E. coli

“…3A). The neck domain mediates multimerization of DC-SIGN, which is required for binding of ligands or pathogens [13,14]. The cytoplasmic tail of DC-SIGN includes internalization motifs, such as the LL and the EEE cluster [2,15], and an incomplete ITAM which is considered to be involved in signal transduction pathway [2,15].…”

“…These results suggest that Ca 2+ binding site in the CRD plays important roles in the surface expression as well as the interaction with Ca 2+ and microbes. It has been demonstrated that the neck domain of murine DC-SIGN responsible for tetramer formation is involved in efficient binding to multivalent ligands [16] and the impairment in tetramer formation results in the impairment of the binding to pathogens/ligands [14]. However, the involvement of Ca 2+ binding site of DC-SIGN in its multimerization still remains unclear.…”

Glycodendritic structures: tools to interact with DC-SIGN

“…It is a type II trans-membrane C-type lectin with a single C-terminal Carbohydrate Recognition Domain (CRD). In the cellular membrane, DC-SIGN is assembled as a tetramer, due to an extended coiled-coil region that allows simultaneous presentation of four CRDs Feinberg et al, 2005;Serrano-Sierra-Gomez et al, 2008;Tabarani et al, 2009).…”